The present invention relates to specific novel organoclay-enzyme compositions, wherein the enzymes are immobilized without inhibiting their activity. The invention relates to an economic method of preparation of enzyme clad organoclay.
Enzymes are known to be active and highly selective catalysts for many applications involving aqueous solutions of substrate compounds. An enzyme composite is rendered insoluble, thus amenable for reuse, when fixed upon a water-insoluble support U.S. Pat. No. 3,953,292 describes how to directly attach enzymes by covalently bonding them to an organic polymeric matrix or to a porous inorganic solid. Particulate insoluble enzymatically active enzymes were produced with a solid siliceous support material including glass. The siliceous support is reacted with certain organosilanes, attaching the enzymes to the reacted organosilanes with a crosslinking agent such as a dialdehyde like glutacaldehyde. In this patent heat activated attapulgite was used as a support. The enzyme was coupled to the attapulgite aggregates by an intermediate silane coupling agent. U.S. Pat. No. 3,669,841 describes a similar method. Clay-enzyme reactions were patented as far back as 1962. U.S. Pat. No. 3,066,026 describes enzyme clad clays used in the brewing of beer.
U.S. Pat. No. 3,650,967 describes dry mixing an enzyme such as protease with a salt such as sodium tripolyphosphate, an organic polymer such as carboxymethyl cellulose, and a clay mineral in a rotating drum. This results in granules which are incorporated into dry laundry detergents. It is not reported whether the enzyme attached to the clay during processing.
U.S. Pat. No. 4,605,621 describes a method of immobilizing enzymes on organoclays to confer stability. In this manner the enzyme is available for the projected use, without its catalytic ability being limited by the immobilization mechanism. This patent reports that the enzymes are bound to the organoclay by some sort of hydrophobic bonding. This method is pH independent. The patent teaches how to prepare an organoclay by mixing a quaternary amine such as hexadecyl trimethyl ammonium bromide with bentonite in a glass jar, including washing of the clay, several hours of equilibration, filtering and washing again with distilled water, followed by freezing and freeze drying. Once the organoclay is dry, the organoclay powder is introduced into a glass jar, where the enzyme powder, urease in this case, was previously dissolved in water at a 1% (w/w) addition level. The authors found that they could add urease at a level as high as 40% by weight of the organoclay, with resulting immobilization. Needless to say, this process is far too complicated and expensive to be put into practice in industry.
U.S. Pat. No. 4,818,695 teaches how microbial lipase derived from the thermophilic mucor species used for the esterification of fats is immobilized on an ion exchange resin. The enzyme powder is dissolved in water, and the resulting slurry is mixed with a particulate macroporous weak anion exchange resin. The resin is then filtered out of the slurry and dried. By providing a specified proportion of water content in the final immobilized preparation, continuous esterification of fats without a solvent is possible. This process results in a strong bond between the lipase and ion exchange resin. However, it takes 5-8 hours of contact time between the resin and the lipase to remove 75% of the enzyme from water and immobilize it on the resin, which is not economical in practice. The resin must then be rigorously washed with water to improve the performance of the final product. Drying of the resin, which must be done down to a moisture content of 20%, is done in a vacuum drying oven, fluid bed or some other drying method. Once the enzyme clad resin is introduced in the esterification process, a continuous, low cost method is at work. However, the preparation of the resin is economically unattractive.
The present invention solves t he problems enumerated above by providing a method for immobilizing enzymes, comprising the step of simultaneously mixing an aqueous enzyme to be immobilized with a quaternary ionic compound and a mineral; wherein the mineral and the quaternary ionic compound react simultaneously with the aqueous enzyme to form an organoclay material having the enzyme immobilized thereon.
Soil eco-enzymes such as urease are immobilized on the organic matter portion of organic mineral complexes in soil. Such complexation confers stability to the urease. When glucose oxidase is immobilized on an organoclay, hydrophobic bonding occurs. The enzyme is strongly bound to the protruding end of the quaternary amine regardless of the pH. The final product exhibits a pH profile similar to the free enzyme (enzyme not immobilized).
Organoclay clad enzymes were prerpared in the following manner: Bentonite was mixed with quaternary amine of the di-methyl dihydrogenated tallow ammonium chloride type surfactant, in a bowl. A small amount of water was also added, and the paste was passed through a laboratory meatgrinder several times to assure thorough mixing. The resulting noodles are then air dried and ground into granules or milled into a free flowing powder.